Transcription-driven DNA supercoiling counteracts H-NS-mediated gene silencing in bacterial chromatin

In all living cells, genomic DNA is compacted through interactions with dedicated proteins and/or the formation of plectonemic coils. In bacteria, DNA compaction is achieved dynamically, coordinated with dense and constantly changing transcriptional activity. H-NS, a major bacterial nucleoid structuring protein, is of special interest due to its interplay with RNA polymerase. H-NS:DNA nucleoprotein filaments inhibit transcription initiation by RNA polymerase. However, the discovery that genes silenced by H-NS can be activated by transcription originating from neighboring regions has suggested that elongating RNA polymerases can disassemble H-NS:DNA filaments. In this study, we present evidence that transcription-induced counter-silencing does not require transcription to reach the silenced gene; rather, it exerts its effect at a distance. Counter-silencing is suppressed by introducing a DNA gyrase binding site within the intervening segment, suggesting that the long-range effect results from transcription-driven positive DNA supercoils diffusing toward the silenced gene. We propose a model wherein H-NS:DNA complexes form in vivo on negatively supercoiled DNA, with H-NS bridging the two arms of the plectoneme. Rotational diffusion of positive supercoils generated by neighboring transcription will cause the H-NS-bound negatively-supercoiled plectoneme to “unroll” disrupting the H-NS bridges and releasing H-NS.

plasmid pNNB5 with primers AG44 and AG45) and subsequently exchanging the cassette with a fragment amplified from plasmid pMP310 (which carries the nuB1 variant of Mu SGS 10 ) with primers AO27-AO28.The tetR-P tet tetA-T hisL cassette was introduced in the SGS background using a recombineering fragment amplified from the MA14400 chromosome with primers AE12-AO31 (see above).Care was taken in designing primer AO31 to ensure that the 3' boundary of the insertion would lie 189 bp closer to hilD than the insertion in the SGS-free context to compensate for the SGS addition (see Supplementary Fig. 5).
When required the tetA ORF was exchanged with the cat ORF using a recombineering fragment amplified from plasmid pKD3 5 with primers AH55 and AN26.
Mutagenesis of the SGS.The segment encompassing the tetRA-T hisL cassette and part of the SGS insert of strain MA14726 (tetR-P tet tetA-T hisL SGS hilA-GFP SF kan ∆K28) was amplified with primers AO37 (Fw primer, annealing near the end of tetR) and AO33 (Rv primer, annealing inside the SGS but with the homology interrupted by a randomized 12-nt stretch -beginning 42 nt from the 5' end of the primer -corresponding to the site of gyrase cleavage).The amplified fragment was introduced into strain MA14733 (tetR-P tet cat-T hisL SGS hilA-GFP SF kan ∆K28 / pKD46) and recombinants were selected on tetracycline-supplemented plates.Colonies showing higher green-fluorescence levels (see Supplementary Fig. 5) were picked and used for PCR and Sanger sequence analysis of the mutagenized region.Performing the same experiment with strain MA14733 as the source of template DNA and strain MA14726 / pKD46 as the recombineering host, allowed isolating SGS mutants in the P tet cat background.

5' RACE-Seq
Experiments.5'RACE-Seq analysis was conducted on three independent RNA preparations from AHTc-treated and untreated cultures of strains MA14692 (∆[sitA-prgH]1.2::tetRA-ThisL ) and MA14606 (leuL::tetRA-T hisL ).RNA was reverse-transcribed with a mixture of primers AI69 (hilD-specific) and AM99 (leuO-specifc) in the presence of AI39, the template switching oligonucleotide (TSO).Each of the cDNAs produced (12 samples) was amplified by PCR with primers carrying adapter sequences designed for high throughput sequencing.Oligonucleotide AK47 (which anneals to the TSO sequence) was the common forward primer in all reactions, while a mixture of two oligonucleotides, one annealing in the promoters-proximal region of hilD, the other annealing in the promoter-proximal region of leuO, and carrying specific index sequences, was used for reverse priming (Supplementary Table 4; amplification program specified in Methods).The PCR products were pooled in equal volumes and the pooled sample was subjected to high throughput sequencing.In the data originating from MA14692, the reads containing the TSO sequence fused to the 5' end of hilD were normalized to the reads with the TSO sequence fused to the 5' end of leuO.Vice versa for the reads originating for MA14606.
In a separate experiment, the RNA preparations from MA14692 were reverse-transcribed with a mixture of primers AI48 (prgH-specific) and AJ33 (ompA-specific) in the presence of the TSO.cDNAs was amplified with AJ38 (which anneals to the TSO sequence) as the common forward primer and a single indexed oligonucleotide specific for either prgH or ompA as the reverse primer.The PCR products were pooled in equal volumes and the pooled sample was subjected to high throughput sequencing.The reads containing the TSO sequence fused to the 5' end of prgH were normalized to the reads with the TSO sequence fused to the 5' end of ompA.

Supplementary Fig. 2
Comparing the effects of ac4va4ng the P tet promoter in constructs without (a) or with (b) the tetA gene fused to P tet .Corresponding pairs of tetR-P tet and tetR-P tet -tetA inserts have iden(cal 3' -flanking sequences.These 3' boundaries fall between 3.0 Kb and 0.6 Kb from the hilD TSS.All strains carry a hilA-GFP SF transla(onal fusion and a cons(tu(vely expressed mCherry fusion to the P tac promoter.Strains were grown at 37°C to early sta(onary phase and cells visualized by fluorescence microscopy under 100 x magnifica(on.Representa(ve areas of the microscope field are shown.